PROJECT SUMMARY/ABSTRACT In this R01 project, we will test a magnetic resonance spectroscopic imaging (MRSI) technique to assess several markers of neuroinflammation across the entire brain. We will use the technique to investigate the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), a condition of unknown etiology that is characterized by profound fatigue not alleviated by rest. The lack of information on ME/CFS pathophysiology has posed a substantial obstacle to the development of treatments that are specific and effective for the disorder. We hypothesize that ME/CFS is the result of low-level inflammation in the brain. Chronic activation of microglia and astrocytes provokes the release of proinflammatory agents that interact with neurons to cause symptoms of fatigue, pain sensitivity, and cognitive and mood disruption. MRSI may be able to detect that neuroinflammation by showing elevated myo-inositol, choline, lactate, brain temperature, and lower N-acetylaspartate that have been associated with abnormal microglia activation. In this five-year R01 study, we will conduct three separate studies. Study #1 examines 90 women with ME/CFS and 30 age- and body mass index-matched healthy controls. Neuroinflammatory markers will be assessed on a voxel-by- voxel basis throughout the entire brain, yielding approximately 4,000 assessments in gray matter, white matter, and cerebrospinal fluid. We hypothesize that the neuroinflammatory markers will be elevated in several brain regions in the ME/CFS group. Study #2 uses a ?good-day, bad-day? longitudinal design to examine correlation between neuroinflammatory markers and symptom severity fluctuations in 20 women with ME/CFS. We hypothesize that the higher fatigue severity days will be associated with higher levels of neuroinflammatory markers. In Study #3, we will validate the MRSI scan with positron emission tomography (PET) analysis of 18F-DPA-714, a marker of activated microglia. We expect to see spatial overlap in MRSI and PET indicators of neuroinflammation. Support for these three hypotheses would show that ME/CFS is associated with brain inflammation. This test would allow for safe and inexpensive longitudinal assessment of neuroinflammation that is not possible with positron emission tomography (PET) or lumbar puncture measures of cerebrospinal fluid. Because we collect the entire available spectrum in each voxel, we will also have the first whole-brain metabolic data in ME/CFS. The MRS data can be used to quantify other markers of interest to ME/CFS researchers, such as glutamate and glutamine. We will therefore make the entire dataset available to other researchers for secondary analyses. Ultimately, we hope this non-invasive scanning technique will aid in ME/CFS diagnosis, treatment decisions, and the development of new treatments.